Graft materials containing bioactive substances, and methods for their manufacture

ABSTRACT

Described are packaged, sterile medical graft products containing controlled levels of a growth factor such as Fibrob-last Growth Factor-2 (FGF-2). Also described are methods of manufacturing medical graft products wherein processing, including sterilization, is controlled and monitored to provide medical graft products having modulated, known levels of a extracellular matrix factor, such as a growth factor, e.g. FGF-2. Preferred graft materials are extracellular matrix materials isolated from human or animal donors, particularly submucosa containing extracellular matrix materials. Further described are ECM compositions that are or are useful for preparing gels, and related methods for preparation and use.

REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. patent application Ser. No.60/497,746 filed Aug. 25, 2003, which is hereby incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates generally to materials useful for tissuegrafting, and in particular to such materials derived from extracellularmatrices and retaining both collagen and substances such as growthfactors that contribute to the beneficial properties of the materials.In one aspect, the invention relates to extracellular matrix tissuegraft materials containing one or more growth factors modulated to apredetermined level, and related methods of manufacturing.

Extracellular matrix (ECM) materials, including those derived fromsubmucosa and other tissues, are known tissue graft materials. See,e.g., U.S. Pat. Nos. 4,902,508, 4,956,178, 5,281,422, 5,372,821,5,554,389, 6,099,567, and 6,206,931. Tissues from various biologicalstructures can be used for these purposes, including for example smallintestine, stomach, the urinary bladder, skin, pericardium, dura mater,fascia, and the like. These sources provide collagenous materials usefulin a variety of surgical procedures where tissue support and/or ingrowthare desired.

Submucosa and other ECM materials have been shown to include a varietyof components other than collagen that that can contribute to thebioactivity of the materials and to their value in medical grafting andother uses. As examples, ECM materials can include growth factors, celladhesion proteins, and proteoglycans. However, ECM materials aretypically subjected to a battery of manipulations in the manufacture offinished products containing them. This presents challenges in obtainingfinished products that not only possess the necessary physicalproperties and appropriate levels of biocompatibility and sterility, butalso the desired bioactivity. The present invention is addressed tothese needs.

SUMMARY OF THE INVENTION

Accordingly, in one aspect, the present invention provides a method formanufacturing a tissue graft material such as a collagenousextracellular matrix containing at least one extractable, bioactivegrowth factor or other non-collagenous protein material, particularlyFibroblast Growth Factor-2 (FGF-2), at a predetermined amount. Themethod includes the steps of providing a non-sterile extracellularmatrix material; fashioning a plurality of graft products from theextracellular matrix material; packaging the products; subjecting thepackaged products to a sterilization procedure that affects the level ofextractable bioactive growth factor (FGF-2) or other non-collagenousprotein material in the products; and, taking and testing sampleproducts of the sterilized packaged products to determine a level of agrowth factor (FGF-2) in the sample products, wherein said determinedlevel is representative of an approximate level of said growth factor inother ones of said products from the lot from which the sample productwas taken.

In another aspect, the present invention provides a medical product thatcomprises a packaged, sterile animal-derived extracellular matrixmaterial comprising FGF-2 at a level of at least about 50 nanograms pergram dry weight. Particularly preferred materials are lyophilized and/orinclude submucosa.

Another aspect of the invention provides a packaged, sterileextracellular matrix material isolated from animal tissue and includingcomponents native to the tissue, the matrix material including collagen,growth factors, proteoglycans, glycosaminoglycans, and havingextractable, bioactive FGF-2 at a level of at least about 50 nanogramsper gram dry weight.

Another aspect of the invention provides a method for manufacturing asterile, extracellular matrix material. The method includes isolating anextracellular matrix material from animal tissue, the isolatedextracellular matrix material including extractable FGF-2 at a firstlevel; and, sterilizing the isolated extracellular matrix material underconditions to retain the extractable, bioactive FGF-2 in at least 10% ofthe first level.

Another aspect of the invention provides a method for manufacturingmedical products. The method includes providing extracellular matrixmaterial in non-sterile condition and isolated from animal tissue, theextracellular matrix material comprising extractable, bioactive FGF-2;packaging and sterilizing the extracellular matrix material to provideproduct lots each containing multiple, packaged extracellular matrixmaterial products; taking sample products from the product lots; andtesting the sample products to determine whether they includeextractable, bioactive FGF-2 at a level above a predetermined level,e.g. above about 50 nanograms per gram dry weight.

Another aspect of the invention provides a medical product adapted fortreating wounds, the product including an extracellular matrix materialisolated from animal tissue, the material including bioactive componentsuseful to treat wounds including but not limited to FGF-2. The FGF-2 ispresent in the extracellular matrix material at a level of at leastabout 50 nanograms per gram dry weight.

Another aspect of the invention relates to a medical product comprisinga dry collagenous powder comprising extracellular matrix material,wherein the dry collagenous powder is effective to gel upon rehydrationwith an aqueous medium and comprises FGF-2 at a level of at least about50 ng/g dry weight.

In another aspect, the invention relates to a medical product comprisinga fluid composition comprising solubilized or suspended collagenousextracellular matrix material, wherein the fluid composition comprisesFGF-2 at a level of about 0.1 ng/ml to about 100 ng/ml.

In another embodiment, the invention provides a method for disinfectingan aqueous extracellular matrix hydrolysate composition. The aqueousextracellular matrix hydrolysate composition is contacted with anoxidizing disinfectant for a period of time and under conditionssufficient to disinfect the aqueous extracellular matrix hydrolysatecomposition.

Another aspect of the invention relates to a method for preparing adisinfected, extracellular matrix hydrolysate composition. This methodcomprises forming an aqueous extracellular matrix hydrolysate. A firstdialysis step is conducted and includes dialyzing the aqueousextracellular matrix hydrolysate against an aqueous medium containing anoxidizing disinfectant so as to contact and disinfect the extracellularmatrix hydrolysate with the oxidizing disinfectant and thereby form adisinfected extracellular matrix hydrolysate. A second dialysis stepincludes dialyzing the disinfected extracellular matrix hydrolysateunder conditions to remove the oxidizing disinfectant.

In another embodiment, the invention provides an extracellular matrixhydrolysate product having extracellular matrix components disinfectedby contact of an aqueous medium containing the extracellular matrixhydrolysate with an oxidizing disinfectant. The extracellular matrixhydrolysate product can take on a variety of forms, including a drypowdery material, a non-gelled aqueous composition, a gel, or a sponge.

Still another embodiment of the invention provides an extracellularmatrix graft material that includes an extracellular matrix hydrolysatecombined with extracellular matrix particles. In a preferred form, thegraft material includes an aqueous medium having said extracellularmatrix hydrolysate in a dissolved state with the extracellular matrixparticles suspended therein, desirably wherein the medium exhibitsgel-forming capacity.

Another embodiment of the invention provides an extracellular matrixgraft material that includes a sterile, injectable fluid extracellularmatrix composition including an aqueous medium containing anextracellular matrix hydrolysate. The extracellular matrix hydrolysateis present in the composition at a level of at least about 20 mg/ml, forexample in the range of about 20 mg/ml to about 200 mg/ml.

Additional aspects as well as features and advantages of the inventionwill be apparent to those of ordinary skill in the art from thedescriptions herein.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of theinvention, reference will now be made to certain embodiments andspecific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of theinvention is thereby intended, such alterations and furthermodifications in the illustrated device, and such further applicationsof the principles of the invention as described herein beingcontemplated as would normally occur to one skilled in the art to whichthe invention relates.

As disclosed above, in one aspect, the present invention providespackaged, sterile medical products including tissue grafts materialscontaining one or more growth factors, and methods for manufacturing thesame. As for the tissue graft material used, it will desirably be anaturally-derived material such as an extracellular matrix (ECM)material. Preferred are naturally-derived collagenous ECMs isolated fromsuitable animal or human tissue sources. Suitable extracellular matrixmaterials include, for instance, submucosa (including for example smallintestinal submucosa, stomach submucosa, urinary bladder submucosa, oruterine submucosa, each of these isolated from juvenile or adultanimals), renal capsule membrane, amnion, dura mater, pericardium,serosa, peritoneum or basement membrane materials, including liverbasement membrane or epithelial basement membrane materials. Thesematerials may be isolated and used as intact natural sheet forms, orreconstituted collagen layers including collagen derived from thesematerials and/or other collagenous materials may be used. For additionalinformation as to submucosa materials useful in the present invention,and their isolation and treatment, reference can be made to U.S. Pat.Nos. 4,902,508, 5,554,389, 5,993,844,6,206,931, and 6,099,567. Renalcapsule membrane can also be obtained from warm-blooded vertebrates, asdescribed more particularly in International Patent Application serialNo. PCT/US02/20499 filed Jun. 28, 2002, published Jan. 9, 2003 asW003002165.

Preferred ECM base materials contain residual bioactive proteins orother ECM components derived from the tissue source of the materials.For example, they may contain Fibroblast Growth Factor-2 (basic FGP),Transforming Growth Factor-beta (TGF-beta) and vascular endothelialgrowth factor (VEGF). It is also expected that ECM base materials of theinvention may contain additional bioactive components including, forexample, one or more of glycosaminoglycans, glycoproteins,proteoglycans, and/or growth factors.

It has been discovered that the sterilization conditions utilized in themanufacture of tissue graft materials can significantly impact the levelof one or more of such bioactive components or growth factors, includingfor example FGF-2. Accordingly, in accordance with the invention,sterilization protocols can be selected and controlled to modulate thelevel of growth factors, for example by either intentionally reducinggrowth factor levels to a predetermined level or below, or to retain atleast a given percentage or level of one or more growth factors,particularly FGF-2, in the material. In certain embodiments of theinvention, the ECM or other graft material is processed to finished,packaged, sterile products containing FGF-2 at a level of at least 50ng/g dry weight, or even at least about 60, at least about 70, at leastabout 80, or at least about 100 ng/g dry weight. In other embodiments ofthe invention, an ECM material will have a first level of a bioactivecomponent, such as FGF-2 or another growth factor, after isolation fromthe animal or human donor source tissue and rinsing with a rinse agentsuch as water. The ECM material will thereafter be processed undercontrolled conditions, including sterilization, to provide packaged,sterile medical products containing at least about 10% of said firstlevel of the FGF-2 or other bioactive component, or even at least 15%,20%, 30% or even 50% or more of said first level.

Illustratively, it has been found that sterilization protocols includingethylene oxide (EO) sterilization, electron beam (E-beam) radiation andgas plasma sterilization (e.g. Sterrad®) can significantly reduce levelsof extractable, bioactive FGF-2. At the same time, these sterilizationtechniques have significantly lower or essentially no impact on levelsof extractable, bioactive TGF-beta. Advantageously, the modulation ofgrowth factors imparted by the sterilization technique can be used toaffect and optimize levels of given growth factors, their ratios, etc.,to prepare a graft material better suited for a particular medicalindication wherein the retained growth factor or growth factors arebeneficial to the indication, and/or wherein eliminated growth factor orgrowth factors are deleterious to the medical indication.

For example, FGF-2 is known to stimulate angiogenesis, neurite growth,plasminogen activator (PA) secretion, and matrix metalloproteinase 1(MMP1) production. Correspondingly, levels of FGF-2 can be retained andoptimized for use in the graft material in wound healing (angiogenesis),treatment of nervous tissue (neurite outgrowth) including peripheralnervous tissue and central nervous tissue, modulating adhesion formation(by stimulating PA), and facilitating collagen turnover and degradation(by stimulating MMP-1 production). Thus, FGF-2 levels can be retained inthe material as high as possible by selecting and optimizing thesterilization protocol. For instance, it has been found that non-sterileisolated submucosa layers (and in particular isolated from smallintestine), contain relatively high levels of extractable, bioactiveFGF-2. For example, submucosa tissue isolated from small intestine andminimally treated, e.g. only by rinsing, may be recovered so as tocontain in excess of about 100 nanograms per gram of FGF-2 dry weightand potentially even higher levels such as above about 200 or about 400nanograms per gram. In manufacturing, it may be beneficial to retain asmuch of this FGF-2 in the material as possible. Thus, intermediate stepsbetween the isolation of the original submucosa material and thefinished, packaged medical article, can be selected and controlled so asto maintain as much active FGF-2 in the material as possible.

As one example, an isolated, small intestinal submucosa materialdisinfected as described in US Pat. No. 6,206,931 with peracetic acidmay contain from about 70 to about 200 nanograms per gram (dry weight)of FGF-2. It has been found that sterilization treatments using ethyleneoxide, E-beam, and gas plasma sterilization techniques significantlyreduce the levels of FGF-2 in the disinfected material. Among these,E-beam sterilization had the smallest impact on FGF-2 levels, withE-beam sterilized submucosa having FGF-2 levels ranging from about 75nanograms per gram dry weight to about 150 nanograms per gram dryweight, and generally retaining greater than about 50% of the FGF-2level of the disinfected submucosa material. Gas plasma sterilizedmaterial had an FGF-2 level ranging from about 60 nanograms per gram dryweight to about 110 nanograms per gram dry weight, and retaining atleast 40% of the FGF-2 level of the disinfected submucosa material.Thus, in embodiments of the invention, materials sterilized using E-beamor gas plasma techniques are used in products configured for and methodsfor treating patients where relatively high FGF-2 levels are beneficial,for example wound healing, treatment of tissue of the nervous system,modulating adhesions, or facilitating collagen turnover and degradation.

On the other hand, ethylene oxide sterilization at both low temperatureand high temperature conditions had a more significant impact inreducing the FGF-2 levels, with products typically having from about 10to about 40 nanograms per gram of FGF-2 dry weight, and retaining lessthan about 40% of the FGF-2 level of the disinfected submucosa material(e.g. about 10% to about 40%). In this ethylene oxide work, the hightemperature conditions tended to do have a slightly greater effect inreducing the FGF-2 levels than the low temperature conditions.Accordingly, in the ethylene oxide and potentially other sterilizationtechniques, the temperature may be increased or decreased to provide arespective higher or lower level of reduction of FGF-2 and/or othergrowth factors or non-collagenous ECM proteins. Similarly, the totaldose of sterilant chemical or energy can be increased or decreased toprovide a respective higher or lower level of reduction of FGF-2 and/orother growth factors or non-collagenous ECM proteins. Increased doses ofsterilant can be achieved, for instance, through a longer, singleexposure of the graft material to the sterilant, or through multiple,discreet exposures of the graft material to the sterilant.

In accordance with the invention, in addition to controlling thesterilization protocol, a number of other manufacturing techniques canbe undertaken to provide a packaged, sterilized graft product with acontrolled level of one or more growth factors, including for exampleFGF-2. As a first measure, where it is desired to retain as high aspossible a level of FGF-2, the animal-derived collagenous ECM can beprocessed and preserved from the time of harvest to the time at whichFGF-2 or other growth factor is protected against further significantdegradation. For these purposes, the harvested tissue from which the ECMmaterial is to be isolated may be placed soon or immediately afterharvest in a stabilizing solution that prevents degradation of theproduct including for example, osmotic, hypoxic, autolytic, and/orproteolytic degradation. This solution can also protect againstbacterial contamination. To achieve these effects, the stabilizingmaterial may be a buffered solution of anti-oxidants, antibiotics,protease inhibitors, oncotic agents, or other stabilizing agents.

Illustratively, enzymes (e.g. superoxide dismutase and catalase) may beused to neutralize the superoxide anion and hydrogen peroxide orcompounds that can directly react with and neutralize other free-radicalspecies. Antioxidants may be added and include tertiarybutylhydroquinone (BHT), alpha tocopherol, mannitol, hydroxyurea,glutathione, ascorbate, ethylenediaminetetraacetic acid (EDTA) and theamino acids histidine, proline and cysteine. In addition toantioxidants, the stabilizing solution may contain agents to inhibithypoxic alteration to normal biochemical pathways, for example,allopurinol to inhibit xanthine dehydrogenase, lipoxigenase inhibitors,calcium channel blocking drugs, calcium binding agents, iron bindingagents, metabolic intermediaries and substrates of adenosinetriphosphate (ATP) generation.

The stabilizing solution may also contain one or more antibiotics,antifungal agents, protease inhibitors, proteoglycans, and anappropriate buffer. Antibiotics can be used to inhibit or preventbacterial growth and subsequent tissue infection. Antibiotics may beselected from the group of penicillin, streptomycin, gentamicin,kanamycin, neomycin, bacitracin, and vancomycin. Additionally,anti-fungal agents may be employed, including amphotericin-B, nystatinand polymyxin.

Protease inhibitors may be included in the stabilizing solution toinhibit endogenous proteolytic enzymes which, when released, can causeirreversible degradation of the ECM, as well as the release ofchemoattractant factors. These chemoattractants solicit the involvementof polymorphonuclear leukocytes, macrophages and other natural killercells which generate a nonspecific immune response that can furtherdamage the ECM. Protease inhibitors can be selected from the groupconsisting of N-ethylmaleimide (NEM), phenylmethylsulfonyl fluoride(PMSF), ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), leupeptin,ammonium chloride, elevated pH and apoprotinin.

Glycosaminoglycans may be included in the stabilizing solution toprovide a colloid osmotic balance between the solution and the tissue,thereby preventing the diffusion of endogenous glycosaminoglycans fromthe tissue to the solution. Endogenous glycosaminoglycans serve avariety of functions in collagen-based connective tissue physiology.They may be involved in the regulation of cell growth anddifferentiation (e.g. heparin sulfate and smooth muscle cells) or,alternatively, they are important in preventing pathologicalcalcification (as with heart valves). Glycosaminoglyeans are alsoinvolved in the complex regulation of collagen and elastin synthesis andremodeling, which is fundamental to connective tissue function.Glycosaminoglycans are selected from the group of chondroitin sulfate,heparin sulfate, and dermatan sulfate and hyaluronan.Non-glycosaminoglycan osmotic agents which may also be included arepolymers such as dextran and polyvinyl pyrolodone (PVP) and amino acidssuch as glycine and proline.

The stabilizing solution can also contain an appropriate buffer. Thenature of the buffer is important in several aspects of the processingtechnique. Crystalloid, low osmotic strength buffers have beenassociated with damage occurring during saphenous vein procurement andwith comeal storage. Optimum pH and buffering capacity against theproducts of hypoxia damage (described below), is essential. In thiscontext the organic and bicarbonate buffers have distinct advantages.(In red cell storage, acetate-citrate buffers with glycine and glucosehave been shown to be effective in prolonging shelf-life and maintainingcellular integrity.) The inventors prefer to use an organic bufferselected from the group consisting of 2-(N-morpholino)ethanesulfonicacid (MES), 3-(N-morpholine)propanesulfonic acid (MOPS) andN-2-hydroxyethylpiperazine-N′2-ethane-sulfonic acid (HEPES).Alternatively, a low salt or physiological buffer, including phosphate,bicarbonate and acetate-citrate, may be more appropriate in certainapplications.

In another aspect, components of the stabilizing solution address one ormore of the events that occur during the harvesting of tissues, such asspasm, hypoxia, hypoxia reperfusion, lysosomal enzyme release, plateletadhesion, sterility and buffering conditions. Involuntary contraction ofthe smooth muscles can result from mechanical stretching or distension,as well as from the chemical action of certain endothelial cell derivedcontraction factors, typically released under hypoxic (low oxygen)conditions. This involuntary contraction may result in damage to theadjacent ECM. For this reason, the stabilizing solution can include oneor more smooth muscle relaxants, selected from the group of calcitoningene related peptide (CGRP), papaverine, sodium nitroprusside (NaNP), H7(a protein Kinase C inhibitor) calcium channel blockers, calciumchelators, isoproterenol, phentolamine, pinacidil,isobutylmethylxanthine (IBMX), nifedepine and flurazine. The harvestedtissue can be immediately placed into this stabilizing solution and ismaintained at 4° C. during transportation and any storage prior tofurther processing.

The tissue graft material of the invention can be provided in anysuitable form, including substantially two-dimensional sheet form(optionally meshed sheet), or a three-dimensional form such as a tube,valve leaflet, or the like. The tissue graft material may contain asingle layer of isolated ECM material, or may be a multilaminateconstruct sized the same as its component layers (e.g. containingdirectly overlapped layers) or larger than its component layers (e.g.containing partially overlapped layers), see, e.g., U.S. Pat. Nos.5,885,619 and 5,711,969.

In another embodiment, the invention provides a medical product thatincludes a dry collagenous powder useful for example to treat wounds orto otherwise induce tissue growth at a desired implant location, andincluding an ECM material. The powder is desirably effective to gel uponrehydration with an aqueous medium and includes FGF-2 at a level of atleast about 50 nanograms per gram dry weight. Illustratively, the powdercan include a particulate of ECM material prepared by drying a fluidizedmaterial prepared as described in U.S. Pat. Nos. 5,516,533 and5,275,826. This resulting powder can be used alone or in combinationwith other powder materials to support gelling of the overall powderupon rehydration with an aqueous medium such as a buffered salinesolution. In this regard, in addition to the particulate ECM material,the powder composition may also include powdered, purified collagen,gelatin, or the like, to assist in gelling the product upon rehydration.

In one embodiment, the preparation of the powder will be conducted toinclude FGF-2 at a level of at least about 50 nanograms per gram dryweight, more preferably at least about 60, 70, 80, or 100 nanograms pergram dry weight. Resultant fluid compositions containing solubilized orsuspended collagenous ECM materials will desirably be prepared tocontain FGF-2 at a level of about 0.1 nanograms per milliliter orgreater, e.g. typically in the range of about 0.1 nanograms per ml toabout 100 nanograms per ml. This fluid composition is desirably gelable,for example upon incubation for a time after rehydration, which may behastened by bringing the fluid composition to a relatively neutral pHand/or to body temperature from room temperature. In other embodiments,the fluidized medical product may contain FGF-2 at a level of about 1 toabout 15 nanograms per ml, or may contain FGF-2 at a level of about 10to about 30 nanograms per ml.

As disclosed above, certain embodiments of the invention providepackaged, sterile medical products. Known packaging techniques andmaterials can be used in the manufacture of such products, with thepackaging being selected to suit the final sterilization technique beingemployed, e.g. ethylene oxide gas, electron-beam, or gas plasmatechniques. In addition, the packaging may contain or otherwise bearindicia relating to the use of the enclosed graft material for aparticular medical indication, e.g. wound care, and/or may contain orotherwise bear indicia as to one or more growth factors (e.g. FGF-2) forwhich the product manufacture has been controlled to modulate its level,e.g. to reflect a minimum level of such growth factor, a maximum levelof such growth factor, or a range of such growth factor contained in theenclosed tissue graft product.

In other embodiments, the present invention provides ECM gelcompositions and methods and materials for their preparation, which canoptionally also be used in conjunction with the techniques describedabove for modulating the level of one or more bioactive substances inthe product, including for example growth factors such as FGF-2. The gelcompositions of the invention can be prepared from an isolated ECMmaterial, for example one of those listed above. The ECM material isused to prepare a solubilized mixture including components of thematerial. This can be achieved by digestion of the ECM material in anacidic or basic medium and/or by contact with an appropriate enzyme orcombination of enzymes.

Typically, the ECM material is reduced to particulate form to aid in thedigestion step. This can be achieved by tearing, cutting, grinding orshearing the isolated ECM material. Illustratively, shearing may beconducted in a fluid medium, and grinding may be conducted with thematerial in a frozen state. For example, the material can be contactedwith liquid nitrogen to freeze it for purposes of facilitating grindinginto powder form. Such techniques can involve freezing and pulverizingsubmucosa under liquid nitrogen in an industrial blender.

Any suitable enzyme may be used for an enzymatic digestion step. Suchenzymes include for example serine proteases, aspartyl proteases, andmatrix metalloproteases. The concentration of the enzyme can be adjustedbased on the specific enzyme used, the amount of submucosa to bedigested, the duration of the digestion, the temperature of thereaction, and the desired properties of the final product. In oneembodiment about 0.1% to about 0.2% of enzyme (pepsin, for example) isused and the digestion is conducted under cooled conditions for a periodof time sufficient to substantially digest the ECM material. Thedigestion can be conducted at any suitable temperature, withtemperatures ranging from 4-37° C. being preferred. Likewise, anysuitable duration of digestion can be used, such durations typicallyfalling in the range of about 2-180 hours. The ratio of theconcentration of ECM material (hydrated) to total enzyme usually rangesfrom about 25 to about 125 and more typically the ratio is about 50, andthe digestion is conducted at 40° C. for 24-72 hours. When an enzyme isused to aid in the digestion, the digestion will be performed at a pH atwhich the enzyme is active and more advantageously at a pH at which theenzyme is optimally active. Illustratively, pepsin exhibits optimalactivity at pH's in the range of about 2-4.

The enzymes or other disruptive agents used to solubilize the ECMmaterial can be removed or inactivated before or during the gellingprocess so as not to compromise gel formation or subsequent gelstability. Also, any disruptive agent, particularly enzymes, that remainpresent and active during storage of the tissue will potentially changethe composition and potentially the gelling characteristics of thesolution. Enzymes, such as pepsin, can be inactivated with proteaseinhibitors, a shift to neutral pH, a drop in temperature below 0° C.,heat inactivation or through the removal of the enzyme by fractionation.A combination of these methods can be utilized to stop digestion of theECM material at a predetermined endpoint, for example the ECM materialcan be immediately frozen and later fractionated to limit digestion.

The ECM material is enzymatically digested for a sufficient time toproduce a hydrolysate of ECM components. The ECM can be treated with oneenzyme or with a mixture of enzymes to hydrolyze the structuralcomponents of the material and prepare a hydrolysate having multiplehydrolyzed components of reduced molecular weight. The length ofdigestion time is varied depending on the application, and the digestioncan be extended to completely solubilize the ECM material. In some modesof operation, the ECM material will be treated sufficiently to partiallysolubilize the material to produce a digest composition comprisinghydrolyzed ECM components and nonhydrolyzed ECM components. The digestcomposition can then optionally be further processed to remove at leastsome of the nonhydrolyzed components. For example, the nonhydrolyzedcomponents can be separated from the hydrolyzed portions bycentrifugation, filtration, or other separation techniques known in theart.

Preferred gel compositions of the present invention are prepared fromenzymatically digested vertebrate ECM material that has beenfractionated under acidic conditions, for example including pH rangingfrom about 2 to less than 7, especially to remove low molecular weightcomponents. Typically, the ECM hydrolysate is fractionated by dialysisagainst a solution or other aqueous medium having an acidic pH, e.g. apH ranging from about 2 to about 5, more desirably greater than 3 andless than 7. In addition to fractionating the hydrolysate under acidicconditions, the ECM hydrolysate is typically fractionated underconditions of low ionic strength with minimal concentrations of saltssuch as those usually found in standard buffers such as PBS (i.e. NaCl,KC1, Na₂HPO₄, or KH₂PO₄) that can pass through the dialysis membrane andinto the hydrolysate. Such fractionation conditions work to reduce theionic strength of the ECM hydrolysate and thereby provide enhanced gelforming characteristics.

The hydrolysate solution produced by enzymatic digestion of the ECMmaterial has a characteristic ratio of protein to carbohydrate. Theratio of protein to carbohydrate in the hydrolysate is determined by theenzyme utilized in the digestion step and by the duration of thedigestion. The ratio may be similar to or may be substantially differentfrom the protein to carbohydrate ratio of the undigested ECM tissue. Forexample, digestion of vertebrate ECM material with a protease such aspepsin, followed by dialysis, will form a fractionated ECM hydrolysatehaving a lower protein to carbohydrate ratio relative to the originalECM material.

In accordance with certain embodiments of the invention, shape retaininggel forms of ECM are prepared from ECM material that has beenenzymatically digested and fractionated under acidic conditions to forman ECM hydrolysate that has a protein to carbohydrate ratio differentthan that of the original ECM material. Such fractionation can beachieved entirely or at least in part by dialysis. The molecular weightcut off of the ECM components to be included in the gel material isselected based on the desired properties of the gel. Typically themolecular weight cutoff of the dialysis membrane (the molecular weightabove which the membrane will prevent passage of molecules) is within inthe range of about 2000 to about 10000 Dalton, and more preferably fromabout 3500 to about 5000 Dalton.

In one embodiment of the invention, apart from the potential removal ofundigested ECM components after the digestion step and any controlledfractionation to remove low molecular weight components as discussedabove, the ECM hydrolysate is processed so as to avoid any substantialfurther physical separation of the ECM components. For example, when amore concentrated ECM hydrolysate material is desired, this can beaccomplished by removing water from the system (e.g. by evaporation orlyophilization) as opposed to using conventional “saltingout”/centrifugation techniques that would demonstrate significantselectivity in precipitating and isolating collagen, leaving behindamounts of other desired ECM components. Thus, in certain embodiments ofthe invention, solubilized ECM components of the ECM hydrolysate remainsubstantially unfractionated, or remain substantially unfractionatedabove a predetermined molecular weight cutoff such as that used in thedialysis membrane, e.g. above a given value in the range of about 2000to 10000 Dalton, more preferably about 3500 to about 5000 Dalton.

Vertebrate ECM material can be stored frozen (e.g. at about −20 to about− 80° C.) in either its solid, comminuted or enzymatically digestedforms prior to formation of the gel compositions of the presentinvention, or the material can be stored after being hydrolyzed andfractionated. The ECM material can be stored in solvents that maintainthe collagen in its native form and solubility. For example, onesuitable storage solvent is 0.01 M acetic acid, however other acids canbe substituted, such as 0.01 N HCl. In accordance with one embodimentthe fractionated ECM hydrolysate is dried (by lyophilization, forexample) and stored in a dehydrated/lyophilized state. The dried formcan be rehydrated and gelled to form a gel of the present invention.

In accordance with one embodiment, the fractionated ECM hydrolysate willexhibit the capacity to gel upon adjusting the pH of a relatively moreacidic aqueous medium containing it to about 5 to about 9, morepreferably about 6.6 to about 8.0, and typically about 7.2 to about 7.8,thus inducing fibrillogenesis and matrix gel assembly. In oneembodiment, the pH of the fractionated hydrolysate is adjusted by theaddition of a buffer that does not leave a toxic residue, and has aphysiological ion concentration and the capacity to hold physiologicalpH. Examples of suitable buffers include PBS, HEPES, and DMEM. In oneembodiment the pH of the fractionated ECM hydrolysate is raised by theaddition of a buffered NaOH solution to 6.6 to 8.0, more preferably 7.2to 7.8. Any suitable concentration of NaOH solution can be used forthese purposes, for example including about 0.05 M to about 0.5 M NaOH.In accordance with one embodiment, the ECM hydrolysate is mixed with abuffer and sufficient 0.25 N NaOH is added to the mixture to achieve thedesired pH. If desired at this point, the resultant mixture can bealiquoted into appropriate forms or into designated cultureware andincubated at 37° C. for 0.5 to 1.5 hours to form an ECM gel.

The ionic strength of the ECM hydrolysate is believed to be important inmaintaining the fibers of collagen in a state that allows forfibrillogenesis and matrix gel assembly upon neutralization of thehydrolysate. Accordingly, if needed, the salt concentration of the ECMhydrolysate material can be reduced prior to neutralization of thehydrolysate. The neutralized hydrolysate can be caused to gel at anysuitable temperature, e.g. ranging from about 4° C. to about 40° C. Thetemperature will typically affect the gelling times, which may rangefrom 5 to 120 minutes at the higher gellation temperatures and 1 to 8hours at the lower gellation temperatures. Typically, the hydrolysatewill be gelled at elevated temperatures to hasten the gelling process,for example at 37° C. In this regard, preferred neutralized ECMhydrolysates will be effective to gel in less than about ninety minutesat 37° C., for example approximately thirty to ninety minutes at 37° C.Alternatively, the gel can be stored at 4° C., and under theseconditions the setting of the gel will be delayed, e.g. for about 3-8hours.

Additional components can be added to the hydrolysate compositionbefore, during or after forming the gel. For example, proteinscarbohydrates, growth factors, therapeutics, bioactive agents, nucleicacids, cells or pharmaceuticals can be added. In certain embodiments,such materials are added prior to formation of the gel. This may beaccomplished for example by forming a dry mixture of a powdered ECMhydrolysate with the additional component(s), and then reconstitutingand gelling the mixture, or by incorporating the additional component(s)into an aqueous, ungelled composition of the ECM hydrolysate before,during (e.g. with) or after addition of the neutralization agent. Inother embodiments, the additional component(s) are added to the formedECM gel, e.g. by infusing or mixing the component(s) into the gel and/orcoating them onto the gel.

In one embodiment of the invention, a particulate ECM material will beadded to the hydrolysate composition, which will then be incorporated inthe formed gel. Such particulate ECM materials can be prepared bycutting, tearing, grinding or otherwise comminuting an ECM startingmaterial. For example, a particulate ECM material having an averageparticle size of about 50 microns to about 500 microns may be includedin the hydrolysate, more preferably about 100 microns to about 400microns. The ECM particulate can be added in any suitable amountrelative to the hydrolysate, with preferred ECM particulate to ECMhydrolysate weight ratios (based on dry solids) being about 0.1:1 toabout 200:1, more preferably in the range of 1:1 to about 100:1. Theinclusion of such ECM particulates in the ultimate gel can serve toprovide additional material that can function to provide bioactivity tothe gel (e.g. itself including FGF-2 and/or other growth factors orbioactive substances as discussed herein) and/or serve as scaffoldingmaterial for tissue ingrowth.

In certain embodiments, an ECM hydrolysate material to be used in tissueaugmentation, e.g. in functional or cosmetic purposes, will incorporatean ECM particulate material. In these embodiments, the ECM particulatematerial can be included at a size and in an amount that effectivelyretains an injectable character to the hydrolysate composition, forexample by injection through a needle having a size in the range of 18to 31 gauge (internal diameters of 0.047 inches to about 0.004 inches).In this fashion, non-invasive procedures for tissue augmentation will beprovided, which in preferred cases will involve the injection of anungelled ECM hydrolysate containing suspended ECM particles at arelatively lower (e.g. room) temperature, which will be promoted to forma gelled composition when injected into a patient and thereby brought tophysiologic temperature (about 37° C.).

In other aspects of the invention, it has been discovered thatprocessing techniques that involve contacting the ECM material with adisinfecting oxidizing agent compound can significantly affect not onlythe concentration of bioactive substances but also the gelling qualityof the collagen molecules. In particular, it has been found thatcontacting an ECM material with an oxidizing agent such as peraceticacid prior to digestion to form the ECM hydrolysate can disrupt orimpair the ability of ECM hydrolysate to form a gel. On the other hand,contacting an aqueous medium including ECM hydrolysate components withan oxidizing disinfectant such as a peroxy compound provides an improvedability to recover a disinfected ECM hydrolysate that exhibits thecapacity to form beneficial gels. In accordance with one embodiment ofthe invention, an aqueous medium containing ECM hydrolysate componentsis disinfected by providing a peroxy disinfectant in the aqueous medium.This is advantageously achieved using dialysis to deliver the peroxydisinfectant into and/or to remove the peroxy disinfectant from theaqueous medium containing the hydrolysate. In one preferred embodiment,the aqueous medium containing the ECM hydrolysate is dialyzed against anaqueous medium containing the peroxy disinfectant to deliver thedisinfectant into contact with the ECM hydrolysate, and then is dialyzedagainst an appropriate aqueous medium (e.g. an acidic aqueous medium) toat least substantially remove the peroxy disinfectant from the ECMhydrolysate. During this dialysis step, the peroxy compound passesthrough the dialysis membrane and into the ECM hydrolysate, and contactsECM components for a sufficient period of time to disinfect the ECMcomponents of the hydrolysate. In this regard, typical contact timeswill range from about 0.5 hours to about 8 hours and more typicallyabout 1 hour to about 4 hours. The period of contact will be sufficientto substantially disinfect the digest, including the removal ofendotoxins and inactivation of virus material present. The removal ofthe peroxy disinfectant by dialysis may likewise be conducted over anysuitable period of time, for example having a duration of about 4 toabout 180 hours, more typically about 24 to about 96 hours. In general,the disinfection step will desirably result in a disinfected ECMhydrolysate composition having sufficiently low levels of endotoxins,viral burdens, and other contaminant materials to render it suitable formedical use. Endotoxin levels below about 2 endotoxin units (EUs) pergram (dry weight) are preferred, more preferably below about 1 EU pergram, as are virus levels below 100 plaque forming units per gram (dryweight), more preferably below 1 plaque forming unit per gram.

In one embodiment, the aqueous ECM hydrolysate composition is asubstantially homogeneous solution during the dialysis step fordelivering the oxidizing disinfectant to the hydrolysate compositionand/or during the dialysis step for removing the oxidizing disinfectantfrom the hydrolysate composition. Alternatively, the aqueous hydrolysatecomposition can include suspended ECM hydrolysate particles, optionallyin combination with some dissolved ECM hydrolysate components, duringeither or both of the oxidizing disinfectant delivery and removal steps.Dialysis processes in which at least some of the ECM hydrolysatecomponents are dissolved during the disinfectant delivery and/or removalsteps are preferred and those in which substantially all of the ECMhydrolysate components are dissolved are more preferred.

The disinfection step can be conducted at any suitable temperature, andwill typically be conducted between 0° C. and 37° C., more typicallybetween about 4° C. and about 15° C. During this step, the concentrationof the ECM hydrolysate solids in the aqueous medium is typically in therange of about 2 mg/ml to about 200 mg/ml, and may vary somewhat throughthe course of the dialysis due to the migration of water through themembrane. In certain embodiments of the invention, a relativelyunconcentrated digest is used, having a starting ECM solids level ofabout 5 mg/ml to about 15 mg/ml. In other embodiments of the invention,a relatively concentrated ECM hydrolysate is used at the start of thedisinfection step, for example having a concentration of at least about20 mg/ml and up to about 200 mg/ml, more preferably at least about 100mg/ml and up to about 200 mg/ml. It has been found that the use ofconcentrated ECM hydrolysates during this disinfection processingresults in an ultimate gel composition having higher gel strength thanthat obtained using similar processing with a lower concentration ECMhydrolysate. Accordingly, processes which involve the removal of amountsof water from the ECM hydrolysate resulting from the digestion prior tothe disinfection processing step are preferred. For example, suchprocesses may include removing only a portion of the water (e.g. about10% to about 98% by weight of the water present) prior to thedialysis/disinfection step, or may include rendering the digest to asolid by drying the material by lyophilization or otherwise,reconstituting the dried material in an aqueous medium, and thentreating that aqueous medium with the dialysis/disinfection step.

Certain impacts of dialysis processing conditions upon ECM hydrolysategels are illustrated in specific work to date described moreparticularly in Examples 2-5 below. Generally, several differentsubmucosa hydrolysates were prepared while varying the acid presentduring pepsin digestion and varying the concentration of ECM hydrolysatepresent during dialysis against a peracetic acid (PAA) solution.Specifically, a first gel (A1) was prepared using 0.5 M acetic acid inthe pepsin digestion solution, and about 5-15 mg/ml ECM hydrolysateduring the PAA disinfection; a second gel (A2) was prepared using 0.5 Macetic acid in the pepsin digestion solution, and about 130-150 mg/mlECM hydrolysate during the PAA disinfection; a third gel (H1) wasprepared using 0.01 M hydrochloric acid in the pepsin digestionsolution, and about 5-15 mg/ml ECM hydrolysate during the PAAdisinfection; and a fourth gel (H2) was prepared using 0.01 Mhydrochloric acid in the pepsin digestion solution, and about 130-150mg/ml ECM hydrolysate during the PAA disinfection. The processed ECMhydrolysates were provided in a solution of 0.1 M HCl at a concentrationof about 30 mg/ml, and then PBS was added and the pH of the mixture wasadjusted to 7.5-7.6 with 0.25 M NaOH to gel the composition. Themechanical properties of the various gels were then assessed. Theresults are summarized in Table 1 below. TABLE 1 Compressive CompressiveGel Modulus (kPa) Strength (kPa A1 1 0.5 A2 7 2 H1 10 3 H2 20 7

As can be seen, the gels prepared using high submucosa hydrolysateconcentrations during the disinfection step (A2,H2) were relativelystronger than those prepared using low submucosa hydrolysateconcentrations (A1, H1). In addition, in cell growth assays, the A2 andH2 gels demonstrated an improved capacity to support the proliferationof primary human dermal fibroblast and primary human bladder smoothmuscle cells as compared to the A1 and H1 gels. In other observations,the gels prepared from ECM hydrolysate materials resultant of HCl/pepsindigestion were relatively stronger than the corresponding gels resultantof acetic acid/pepsin digestion. Thus, the conditions used during thepreparation and processing of ECM hydrolysate materials can be selectedand controlled to modulate the physical and biological properties of theultimate ECM gel compositions.

In one mode of operation, the disinfection of the aqueous mediumcontaining the ECM hydrolysate can include adding the peroxy compound orother oxidizing disinfectant directly to the ECM hydrolysate, forexample being included in an aqueous medium used to reconstitute a driedECM hydrolysate or being added directly to an aqueous ECM hydrolysatecomposition. The disinfectant can then be allowed to contact the ECMhydrolysate for a sufficient period of time under suitable conditions(e.g. as described above) to disinfect the hydrolysate, and then removedfrom contact with the hydrolysate. In one embodiment, the oxidizingdisinfectant can then be removed using a dialysis procedure as discussedabove. In other embodiments, the disinfectant can be partially orcompletely removed using other techniques such as chromatographic or ionexchange techniques, or can be partially or completely decomposed tophysiologically acceptable components. For example, when using anoxidizing disinfectant containing hydrogen peroxide (e.g. hydrogenperoxide alone or a peracid such as peracetic acid), hydrogen peroxidecan be allowed or caused to decompose to water and oxygen, for examplein some embodiments including the use of agents that promote thedecomposition such as thermal energy or ionizing radiation, e.g.ultraviolet radiation.

In another mode of operation, the oxidizing disinfectant can bedelivered into the aqueous medium containing the ECM hydrolysate bydialysis and processed sufficiently to disinfect the hydrolysate (e.g.as described above), and then removed using other techniques such aschromatographic or ion exchange techniques in whole or in part, orallowed or caused to decompose in whole or in part as discussedimmediately above.

Peroxygen compounds that may be used in the disinfection step include,for example, hydrogen peroxide, organic peroxy compounds, and preferablyperacids. Such disinfecting agents are used in a liquid medium,preferably a solution, having a pH of about 1.5 to about 10.0, moredesirably about 2.0 to about 6.0. As to peracid compounds that can beused, these include peracetic acid, perpropioic acid, or perbenzoicacid. Peracetic acid is the most preferred disinfecting agent forpurposes of the present invention.

When used, peracetic acid is desirably diluted into about a 2% to about50% by volume of alcohol solution, perferably ethanol. The concentrationof the peracetic acid may range, for instance, from about 0.05% byvolume to about 1.0% by volume. Most preferably, the concentration ofthe peracetic acid is from about 0.1% to about 0.3% by volume. Whenhydrogen peroxide is used, the concentration can range from about 0.05%to about 30% by volume. More desirably the hydrogen peroxideconcentration is from about 1% to about 10% by volume, and mostpreferably from about 2% to about 5% by volume. The solution may or maynot be buffered to a pH from about 5 to about 9, with more preferredpH's being from about 6 to about 7.5. These concentrations of hydrogenperoxide can be diluted in water or in an aqueous solution of about 2%to about 50% by volume of alcohol, most preferably ethanol. Additionalinformation concerning preferred peroxy disinfecting agents can be foundin discussions in U.S. Pat. No. 6,206,931, which is herein incorporatedby reference.

ECM gel materials of the present invention can be prepared to havedesirable properties for handling and use. For example, fluidized ECMhydrolysates can be prepared in an aqueous medium, which can thereafterbe caused or allowed to form of a gel. Such prepared aqueous mediums canhave any suitable level of ECM hydrolysate therein for subsequent gelformation. Typically, the ECM hydrolysate will be present in the aqueousmedium to be gelled at a concentration of about 2 mg/ml to about 200mg/ml, more typically about 20 mg/ml to about 200 mg/ml, and in somepreferred embodiments about 30 mg/ml to about 120 mg/ml. In preferredforms, the aqueous ECM hydrolysate composition to be gelled will have aninjectable character, for example by injection through a needle having asize in the range of 18 to 31 gauge (internal diameters of about 0.047inches to about 0.004 inches).

Furthermore, gel compositions can be prepared so that in addition toneutralization, heating to physiologic temperatures (such as 37° C.)will substantially reduce the gelling time of the material. As well,once the material is gelled, it can optionally be dried to form a spongesolid material. It is contemplated that commercial products mayconstitute any of the these forms of the ECM gel composition, e.g. (i)packaged, sterile powders which can be reconstituted in an acidic mediumand neutralized and potentially heated to form a gel, (ii) packaged,sterile aqueous compositions including solubilized ECM hydrolysatecomponents under non-gelling (e.g. acidic) conditions; (iii) packaged,sterile gel compositions, and (iv) packaged, sterile, dried spongecompositions; or other suitable forms. In one embodiment of theinvention, a medical kit is provided that includes a packaged, sterileaqueous composition including solubilized ECM hydrolysate componentsunder non-gelling (e.g. acidic) conditions, and a separately packaged,sterile aqueous neutralizing composition (e.g. containing a bufferand/or base) that is adapted to neutralize the ECM hydrolysate mediumfor the formation of a gel. In another embodiment of the invention, amedical kit includes a packaged, sterile, dried (e.g. lyophilized) ECMhydrolysate powder, a separately packaged, sterile aqueous acidicreconstituting medium, and a separately packaged sterile, aqueousneutralizing medium. In use, the ECM hydrolysate powder can bereconstituted with the reconstituting medium to form a non-gelledmixture, which can then be neutralized with the neutralizing medium forthe formation of the gel.

Medical kits as described above may also include a device, such as asyringe, for delivering the neutralized ECM hydrolysate medium to apatient. In this regard, the sterile, aqueous ECM hydrolysate medium orthe sterile ECM hydrolysate powder of such kits can be provided packagedin a syringe or other delivery instrument. In addition, the sterilereconstituting and/or neutralizing medium can be packaged in a syringe,and means provided for delivering the contents of the syringe into toanother syringe containing the aqueous ECM hydrolysate medium or the ECMhydrolysate powder for mixing purposes. In still other forms of theinvention, a self-gelling aqueous ECM hydrolysate composition can bepackaged in a container (e.g. a syringe) and stable against gelformation during storage. For example, gel formation of such productscan be dependent upon physical conditions such as temperature or contactwith local milieu present at an implantation site in a patient.Illustratively, an aqueous ECM hydrolysate composition that does not gelor gels only very slowly at temperatures below physiologic temperature(about 37° C.) can be packaged in a syringe or other container andpotentially cooled (including for example frozen) prior to use forinjection or other implantation into a patient.

In particular applications, ECM hydrolysate compositions that formhydrogels at or near physiologic pH and temperature will be preferredfor in vivo bulking applications, for example in the treatment of stressurinary incontinence, gastroesophageal reflux disease, cosmetic surgery,vesico urethral reflux, anal incontinence and vocal cord repair. Theseforms of the submucosa or other ECM gel have, in addition to collagen,complex extracellular matrix sugars and varying amounts of growthfactors in other bioactive agents that can serve to remodel tissue atthe site of implantation. These ECM hydrolysate compositions can, forexample, be injected into a patient for these applications.

ECM gels and dry sponge form materials of the invention prepared bydrying ECM gels can be used, for example, in wound healing and/or tissuereconstructive applications, or in the culture of cells.

Generally, it has been found that the manipulations used to prepare ECMhydrolysate compositions and gellable or gelled forms thereof can alsohave a significant impact upon growth factors or other ECM componentsthat may contribute to bioactivity. Techniques for modulating andsampling for levels of FGF-2 or other growth factors or bioactivesubstances can also be used in conjunction with the manufacture of thedescribed ECM hydrolysate compositions of the invention. Illustratively,it has been discovered that the dialysis/disinfection processes of theinvention employing peroxy compounds typically cause a reduction in thelevel of FGF-2 in the ECM hydrolysate material. In work to date asdescribed in Examples 2-5, such processing using peracetic acid asdisinfectant has caused a reduction in the level of FGF-2 in the rangeof about 30% to about 50%. Accordingly, to retain higher levels ofFGF-2, one can process for a minimal about of time necessary to achievethe desired disinfection of the material; on the other hand, to reducethe FGF-2 to lower levels, the disinfection processing can be continuedfor a longer period of time. In one embodiment of the invention, thedisinfection process and subsequent steps will be sufficiently conductedto result in a medically sterile aqueous ECM hydrolysate composition,which can be packaged using sterile filling operations. In otherembodiments, any terminal sterilization applied to the ECM hydrolysatematerial (e.g. in dried powder, non-gelled aqueous medium, gelled orsponge form) can also be selected and controlled to optimize the levelof FGF-2 or other bioactive substances in the product. Terminalsterilization methods may include, for example, high or low temperatureethylene oxide, radiation such as E-beam, gas plasma (e.g. Sterrad), orhydrogen peroxide vapor processing.

Preferred, packaged, sterilized ECM hydrolysate products prepared inaccordance with the invention will have an FGF-2 level (this FGF-2 beingprovided by the ECM hydrolysate) of about 100 ng/g to about 5000 ng/gbased upon the dry weight of the ECM hydrolysate. More preferably, thisvalue will be about 300 ng/g to about 4000 ng/g. As will be understood,such FGF-2 levels can be determined using standard ELISA tests (e.g.using the Quantikine Human Basic Fibroblast Growth Factor ELISA kitcommercially available from R&D Systems).

In order to promote a further understanding of the present invention andits features and advantages, the following specific examples areprovided. It will be understood that these examples are illustrative andare not limiting of the invention.

EXAMPLE 1

Small intestinal submucosa material was harvested and disinfected withperacetic acid as described in U.S. Pat. No. 6,206,931. The submucosamaterial was lyophilized, packaged in medical packaging comprised ofpolyester/Tyvek and sterilized by various methods including ethyleneoxide (EO), gas plasma (hydrogen peroxide vapor), and E-beam radiation(20 kGy (plus/minus 2 kGy). The resultant submucosa material was frozenin liquid nitrogen and ground to a powder. The material was thenextracted with an extraction buffer containing 2M urea, 2.5 mg/mlheparin, and 50 mM Tris buffer, at pH 7.5 at 4° C. under constantstirring for 24 hours. After 24 hours, the extraction medium wastransferred to centrifuge tubes and the insoluble fraction pelleted at12000×G. The supernatant was transferred to dialysis tubing (MW cutoff3500) and dialyzed exhaustively against high purity (18 megaohm) water.Following dialysis the dialysate was centrifuged at 12000×G to removeany additional particulate matter and the resulting soluble extract waslyophilized. Prior to measurement the extract was reconstituted at 10mg/dry weight per ml in the manufacturer-provided diluent (R&D Systems).Samples were centrifuged to remove any insoluble matter. The resultingsupernatents were recovered and assayed for FGF-2 content using theQuantikine Human Basic Fibroblast Growth Factor Immunoassay (R&DSystems). The results are summarized in Table 2 below. TABLE 2 ELISASummary - CBI Extracted Tissues Growth factor Sterilization range (ng/g)% of Non-sterile* NONE 100-210 100 EO (low temp) 28-50 24 EO (high temp)18-40 18 E-beam  50-150 66 Gas Plasma  30-125 49*based upon the average of 8 experiments.

As can be seen, E-beam and gas plasma sterilization had a significantlylower impact in reducing the level of extractable, bioactive FGF-2 inthe materials. On the other hand, ethylene oxide sterilization at bothlow and high temperatures had a significant impact in lowering the levelof extractable, bioactive FGF-2.

EXAMPLE 2

Raw (isolated/washed but non-disinfected) porcine small intestinesubmucosa was frozen, cut into pieces, and cryoground to powder withliquid nitrogen. 50 g of the submucosa powder was mixed with one literof a digestion solution containing 1 g of pepsin and 0.5 M acetic acid.The digestion process was allowed to continue for 48-72 hours underconstant stirring at 4° C. At the end of the process, the digest wascentrifuged to remove undigested material. The acetic acid was thenremoved by dialysis against 0.01 M HCl for approximately 96 hours at 4°C. The resulting digest was transferred (without concentration) into asemipermeable membrane with a molecular weight cut off of 3500, anddialyzed for two hours against a 0.2 percent by volume peracetic acid ina 5 percent by volume aqueous ethanol solution at 4° C. This step servedboth to disinfect the submucosa digest and to fractionate the digest toremove components with molecular weights below 3500. The PAA-treateddigest was then dialysed against 0.01 M HCl for 48 hours at 4° C. toremove the peracetic acid. The sterilized digest was concentrated bylyophilization, forming a material that was reconstituted at about 30mg/ml solids in 0.01 M HCl and neutralized with phosphate buffered NaOHto a pH of about 7.5-7.6 and heated to physiologic temperature to form asubmucosa gel.

EXAMPLE 3

A second acetic acid processed submucosa gel was made using a processsimilar to that described in Example 2 above, except concentrating thedigest prior to the PAA treatment. Specifically, immediately followingthe removal of acetic acid by dialysis, the digest was lyophilized todryness. A concentrated paste of the digest was made by dissolving apre-weighed amount of the lyophilized product in a known amount of 0.01M HC1 to prepare a mixture having an ECM solids concentration of about50 mg/ml. The concentrated paste was then dialysed against the PAAsolution for 2 hours and then against 0.01 M HCl for removal of PAA inthe same manner described in Example 2. The digest was adjusted to about30 mg/ml solids and neutralized with phosphate buffered NaOH to a pH ofabout 7.5-7.6 and heated to physiologic temperature to form a submucosagel.

EXAMPLE 4

An HCI processed submucosa gel was made using a procedure similar tothat described in Example 2, except using 0.01 M of HCl in thepepsin/digestion solution rather than the 0.5 M of acetic acid, andomitting the step involving removal of acetic acid since none waspresent. The digest was used to form a gel as described in Example 2.

EXAMPLE 5

Another HCI processed submucosa gel was made using a procedure similarto that described in Example 3, except using 0.01 M of HCl in thepepsin/digestion solution rather than the 0.5 M of acetic acid, andomitting the step involving removal of acetic acid since none waspresent. The digest was used to form a gel as described in Example 3.

While the invention has been illustrated and described in detail in thedrawings and foregoing description, the same is to be considered asillustrative and not restrictive in character, it being understood thatonly the preferred embodiment has been shown and described and that allchanges and modifications that come within the spirit of the inventionare desired to be protected. In addition, all publications cited in thisapplication are indicative of the abilities possessed by those ofordinary skill in the pertinent art and are hereby incorporated byreference in their entirety as if each had been individuallyincorporated by reference and fully set forth.

1. A medical product, comprising a packaged, sterile animal-derivedextracellular matrix material comprising extractable, bioactiveFibroblast Growth Factor-2 (FGF-2) at a level of at least about 50 ng/gdry weight.
 2. The medical product of claim 1, wherein the extracellularmatrix material comprises submucosa.
 3. The medical product of claim 2,wherein the submucosa is small intestinal submucosa.
 4. The medicalproduct of claim 2, wherein the submucosa is porcine.
 5. The medicalproduct of claim 1, wherein the FGF-2 is present at a level of at leastabout 60 ng/g dry weight.
 6. The medical product of claim 1, wherein theFGF-2 is present at a level of at least about 70 ng/g dry weight.
 7. Themedical product of claim 1, wherein the FGF-2 is present at a level ofat least about 80 ng/g dry weight.
 8. The medical product of claim 1,wherein the FGF-2 is present at a level of at least about 100 ng/g dryweight.
 9. The medical product of claims1, wherein the extracellularmatrix material has been sterilized with radiation.
 10. The medicalproduct of claim 9, wherein the material has been sterilized with e-beamradiation.
 11. A medical product, comprising a packaged, sterileextracellular matrix material isolated from animal tissue and comprisingcomponents native to said tissue including collagen, growth factors,proteoglycans, and glycosaminoglycans, and having extractable, bioactiveFibroblast Growth Factor-2 (FGF-2) at a level of at least about 50 ng/gdry weight.
 12. The medical product of claim 11, wherein theextracellular matrix material comprises submucosa.
 13. The medicalproduct of claim 12, wherein the submucosa is small intestinalsubmucosa. 14-20. (canceled)
 21. A method for manufacturing a sterile,extracellular matrix material, comprising: isolating an extracellularmatrix material from animal tissue, the isolated extracellular matrixmaterial comprising a first level of extractable FGF-2; processing theisolated material to a sterilized extracellular matrix material underconditions to retain said extractable FGF in at least 10% of said firstlevel.
 22. The method of claim 21, wherein the extracellular matrixmaterial comprises submucosa.
 23. The method of claim 22, wherein thesubmucosa is small intestinal submucosa.
 24. The method of claim 22,wherein the submucosa is porcine. 25-27. (canceled)
 28. The method ofclaim 21, wherein the FGF-2 is present in said sterilized material at alevel of at least about 100 ng/g dry weight.
 29. The method of claim 21,wherein the extracellular matrix material has been sterilized withradiation.
 30. The method of claim 29, wherein the material has beensterilized with e-beam radiation.
 31. A method for manufacturing medicalproducts, comprising: providing extracellular matrix material isolatedfrom animal tissue, the extracellular matrix material comprisingextractable, bioactive FGF-2 at a first level; processing theextracellular matrix material to provide product lots each containingmultiple packaged, sterile extracellular matrix material products;taking sample products from said product lots; and testing said sampleproducts to determine whether they include extractable, bioactive FGF-2at a level of at least about 10% of said first level.
 32. The method ofclaim 31, wherein the extracellular matrix material comprises submucosa.33. The method of claim 32, wherein the submucosa is small intestinalsubmucosa.
 34. The method of claim 32, wherein the submucosa is porcine.35. The method of claim 31, wherein said testing comprises testing forFGF-2 at a level of at least about 50 ng/g dry weight.
 36. The method ofclaim 31, wherein said testing comprises testing said sample products todetermine whether they include extractable, bioactive FGF-2 at a levelof at least about 15% of said first level.
 37. The method of claim 31,wherein said testing comprises testing said sample products to determinewhether they include extractable, bioactive FGF-2 at a level of at leastabout 20% of said first level.
 38. The method of claim 31, wherein saidtesting comprises testing said sample products to determine whether theyinclude extractable, bioactive FGF-2 at a level of at least about 30% ofsaid first level.
 39. The method of claim 31, wherein said testingcomprises testing said sample products to determine whether they includeextractable, bioactive FGF-2 at a level of at least about 50% of saidfirst level.
 40. The method of claim 31, wherein the extracellularmatrix material has been sterilized with e-beam radiation.
 41. A medicalproduct adapted for treating wounds, comprising: an extracellular matrixmaterial isolated from animal tissue, said material including bioactivecomponents useful to treat wounds including FGF-2, said FGF-2 present insaid extracellular matrix material at a level of at least about 50 ng/gdry weight.
 42. The medical product of claim 41, wherein theextracellular matrix material comprises submucosa.
 43. The medicalproduct of claim 42, wherein the submucosa is small intestinalsubmucosa. 44-47. (canceled)
 48. The medical product of claim 41,wherein the FGF-2 is present at a level of at least about 100 ng/g dryweight.
 49. The medical product of claim 41, wherein the extracellularmatrix material has been sterilized with radiation.
 50. The medicalproduct of claim 49, wherein the material has been sterilized withe-beam radiation.
 51. A medical product, comprising: a dry collagenouspowder comprising extracellular matrix material; said dry collagenouspowder effective to gel upon rehydration with an aqueous medium; andsaid dry collagenous powder comprising FGF-2 at a level of at leastabout 50 ng/g dry weight.
 52. The medical product of claim 51, whereinthe extracellular matrix material comprises submucosa.
 53. The medicalproduct of claim 52, wherein the submucosa is small intestinalsubmucosa. 54-57. (canceled)
 58. The medical product of claim 51,wherein the FGF-2 is present at a level of at least about 100 ng/g dryweight.
 59. The medical product of claim 51, wherein the extracellularmatrix material has been sterilized with radiation.
 60. The medicalproduct of claim 59, wherein the material has been sterilized withe-beam radiation.
 61. A medical product, comprising: a fluid compositioncomprising solubilized or suspended collagenous extracellular matrixmaterial; said fluid composition comprising FGF-2 at a level of about0.1 ng/ml to about 100 ng/ml.
 62. The medical product of claim 61,wherein said fluid composition is a gelable composition.
 63. The medicalproduct of claim 61, comprising FGF-2 at a level of about 1 to about 50ng/ml.
 64. The medical product of claim 61, comprising FGF-2 at a levelof about 10 to about 30 ng/ml.
 65. A method for preparing a disinfected,extracellular matrix hydrolysate composition, the method comprising:forming an aqueous extracellular matrix hydrolysate containingsolubilized ECM components; a first dialysis step including dialyzingsaid aqueous extracellular matrix hydrolysate against an aqueous acidicmedium containing an oxidizing disinfectant comprising a peroxy compoundso as to contact and disinfect the extracellular matrix hydrolysate withthe oxidizing disinfectant and thereby form a disinfected extracellularmatrix hydrolysate; and a second dialysis step including dialyzing thedisinfected extracellular matrix hydrolysate under conditions to removethe oxidizing disinfectant.
 66. The method of claim 65, wherein theoxidizing disinfectant comprises an organic peroxy compound.
 67. Themethod of claim 65, wherein the peroxy compound is a peracid.
 68. Themethod of claim 67, wherein the peroxy compound is peracetic acid.69-71. (canceled)
 72. The method of claim 65, wherein the aqueous ECMhydrolysate is concentrated prior to said first dialysis step.
 73. Themethod of claim 72, wherein the aqueous ECM hydrolysate is dried andreconstituted in an aqueous medium prior to said first dialysis step.74. The method of claim 72, wherein the aqueous ECM hydrolysate ispartially dewatered to form a more concentrated aqueous ECM hydrolysateprior to said first dialysis step.
 75. A method for disinfecting anaqueous extracellular matrix hydrolysate composition containingsolubilized extracellular matrix components, comprising adding anoxidizing disinfectant directly to the aqueous extracellular matrixhydrolysate composition and contacting the aqueous extracellular matrixhydrolysate composition with the oxidizing disinfectant for a period oftime and under conditions sufficient to disinfect the aqueousextracellular matrix hydrolysate composition.
 76. The method of claim75, wherein the oxidizing disinfectant comprises peracetic acid.
 77. Themethod of claim 75, also including removing the oxidizing disinfectantfrom the aqueous extracellular matrix hydrolysate composition.
 78. Themethod of claim 75, wherein the extracellular matrix comprisesvertebrate submucosa.
 79. An extracellular matrix hydrolysatecomposition having extracellular matrix components disinfected bycontact in solution with an oxidizing disinfectant comprising a peroxycompound.
 80. The composition of claim 79 wherein the oxidizingdisinfectant comprises a peracid.
 81. The composition of claim 80wherein the oxidizing disinfectant comprises peracetic acid. 82-88.(canceled)
 89. An extracellular matrix graft material, comprising: anextracellular matrix hydrolysate; and extracellular matrix particles.90. The graft material of claim 89, comprising an aqueous mediumincluding said extracellular matrix hydrolysate in a dissolved statewith said extracellular matrix particles suspended therein.
 91. Thegraft material of claim 90 in injectable form.
 92. The graft material ofclaim 89 wherein said extracellular matrix hydrolysate comprises asubmucosa hydrolysate and said extracellular matrix particles comprisesubmucosa particles.
 93. An extracellular matrix graft material,comprising: a sterile, injectable fluid extracellular matrix compositionincluding an aqueous medium containing an extracellular matrixhydrolysate, said extracellular matrix hydrolysate present in saidcomposition at a level of about 20 mg/ml to about 200 mg/ml.
 94. Thegraft material of claim 93 wherein the extracellular matrix hydrolysatecomprises a submucosa hydrolysate.
 95. The graft material of claim 93,wherein said extracellular matrix hydrolysate is present at a level ofabout 30 mg/ml to about 120 mg/ml.
 96. The graft material of claim 93,also including extracellular matrix particles having an average particlesize in the range of about 50 microns to about 500 microns.
 97. Thegraft material of claim 96, wherein the extracellular matrix particlesare included in a weight ratio of about 1:1 to about 100:1 relative tothe extracellular matrix hydrolysate on a dry weight basis.
 98. A methodfor making a disinfected composition comprising an extracellular matrixhydrolysate, comprising: adding an oxidizing disinfectant directly to acomposition comprising an extracellular matrix hydrolysate so as todisinfect the extracellular matrix hydrolysate; and allowing or causingthe added oxidizing disinfectant to decompose in the composition. 99.The method of claim 98, wherein the oxidizing disinfectant compriseshydrogen peroxide.
 100. The method of claim 98, wherein the oxidizingdisinfectant comprises peracetic acid.
 101. An injectable tissuegrafting composition, comprising: a sterile, injectable extracellularmatrix composition; wherein said extracellular matrix compositionincludes an aqueous gel containing an extracellular matrix hydrolysatematerial present in said extracellular matrix composition at a level ofabout 20 mg/ml to about 200 mg/ml; wherein said extracellular matrixcomposition also includes extracellular matrix particles suspended insaid aqueous gel, said extracellular matrix particles having an averageparticle size in the range of about 50 microns to about 500 microns; andwherein said extracellular matrix particles are included at a size andin an amount wherein the extracellular matrix composition isadministrable by injection through a needle having an internal diameterof about 0.047 inches to about 0.004 inches.
 102. The injectable tissuegrafting composition of claim 101, wherein the extracellular matrixcomposition comprises at least one material selected from the groupconsisting of submucosa, renal capsule membrane, amnion, dura mater,pericardium, serosa, peritoneum and basement membrane.
 103. A medicalproduct, comprising a packaged, extracellular matrix material derivedfrom animal tissue that has been subjected to processing to provide amedical tissue grafting material, wherein said processing has retainedin said extracellular matrix material growth factors, proteoglycans andglycosaminoglycans from the animal tissue, wherein said processing hasincluded sterilization by exposure to radiation or gas plasma, andwherein said processing has retained in said extracellular matrixmaterial extractable, bioactive Fibroblast Growth Factor-2 (FGF-2) at alevel of at least about 50 ng/g dry weight.
 104. The medical product ofclaim 103, wherein said processing has included sterilization byexposure to e-beam radiation.
 105. The medical product of claim 103,wherein said processing has included sterilization by exposure to gasplasma.